Methods of sustaining culture viability

ABSTRACT

The present invention relates to methods for sustaining a microbial culture during periods of limited substrate supply. In accordance with the methods of the invention a microbial culture comprising carboxydotrophic bacteria can be sustained during periods of limited substrate supply by maintaining the temperature of the microbial culture at a temperature below an optimum operating temperature.

FIELD OF THE INVENTION

This invention relates generally to methods for increasing theefficiency of microbial growth and production of products by microbialfermentation on gaseous substrates. More particularly the inventionrelates to processes for producing products such as alcohols bymicrobial fermentation before, during and/or after a substrate streamcomprising CO becomes limited. In particular embodiments, the inventionrelates to methods of sustaining viability of a microbial culture duringperiods of limited substrate comprising CO.

BACKGROUND OF THE INVENTION

Ethanol is rapidly becoming a major hydrogen-rich liquid transport fuelaround the world. Worldwide consumption of ethanol in 2005 was anestimated 12.2 billion gallons. The global market for the fuel ethanolindustry has also been predicted to continue to grow sharply in future,due to an increased interest in ethanol in Europe, Japan, the USA andseveral developing nations.

For example, in the USA, ethanol is used to produce E10, a 10% mixtureof ethanol in gasoline. In E10 blends, the ethanol component acts as anoxygenating agent, improving the efficiency of combustion and reducingthe production of air pollutants. In Brazil, ethanol satisfiesapproximately 30% of the transport fuel demand, as both an oxygenatingagent blended in gasoline, and as a pure fuel in its own right. Also, inEurope, environmental concerns surrounding the consequences of GreenHouse Gas (GHG) emissions have been the stimulus for the European Union(EU) to set member nations a mandated target for the consumption ofsustainable transport fuels such as biomass derived ethanol.

The vast majority of fuel ethanol is produced via traditionalyeast-based fermentation processes that use crop derived carbohydrates,such as sucrose extracted from sugarcane or starch extracted from graincrops, as the main carbon source. However, the cost of thesecarbohydrate feed stocks is influenced by their value as human food oranimal feed, and the cultivation of starch or sucrose-producing cropsfor ethanol production is not economically sustainable in allgeographies. Therefore, it is of interest to develop technologies toconvert lower cost and/or more abundant carbon resources into fuelethanol.

CO is a major, free, energy-rich by-product of the incomplete combustionof organic materials such as coal or oil and oil derived products. Forexample, the steel industry in Australia is reported to produce andrelease into the atmosphere over 500,000 tonnes of CO annually.

Catalytic processes may be used to convert gases consisting primarily ofCO and/or CO and hydrogen (H₂) into a variety of fuels and chemicals.Micro-organisms may also be used to convert these gases into fuels andchemicals. These biological processes, although generally slower thanchemical reactions, have several advantages over catalytic processes,including higher specificity, higher yields, lower energy costs andgreater resistance to poisoning.

The ability of micro-organisms to grow on CO as a sole carbon source wasfirst discovered in 1903. This was later determined to be a property oforganisms that use the acetyl coenzyme A (acetyl CoA) biochemicalpathway of autotrophic growth (also known as the Woods-Ljungdahl pathwayand the carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS)pathway). A large number of anaerobic organisms includingcarboxydotrophic, photosynthetic, methanogenic and acetogenic organismshave been shown to metabolize CO to various end products, namely CO₂,H₂, methane, n-butanol, acetate and ethanol. While using CO as the solecarbon source, all such organisms produce at least two of these endproducts.

Anaerobic bacteria, such as those from the genus Clostridium, have beendemonstrated to produce ethanol from CO, CO₂ and H₂ via the acetyl CoAbiochemical pathway. For example, various strains of ClostridiumIjungdahlii that produce ethanol from gases are described in WO00/68407, EP 117309, U.S. Pat. Nos. 5,173,429, 5,593,886, and 6,368,819,WO 98/00558 and WO 02/08438. The bacterium Clostridium autoethanogenumsp is also known to produce ethanol from gases (Abrini et al., Archivesof Microbiology 161, pp 345-351 (1994)).

However, ethanol production by micro-organisms by fermentation of gasesis always associated with co-production of acetate and/or acetic acid.As some of the available carbon is converted into acetate/acetic acidrather than ethanol, the efficiency of production of ethanol using suchfermentation processes may be less than desirable. Also, unless theacetate/acetic acid by-product can be used for some other purpose, itmay pose a waste disposal problem. Acetate/acetic acid is converted tomethane by micro-organisms and therefore has the potential to contributeto GHG emissions.

Several enzymes known to be associated with the ability ofmicro-organisms to use carbon monoxide as their sole source of carbonand energy are known to require metal co-factors for their activity.Examples of key enzymes requiring metal cofactor binding for activityinclude carbon monoxide dehydrogenase (CODH), and acetyl —CoA synthase(ACS).

WO2007/117157 and WO2008/115080, the disclosure of which areincorporated herein by reference, describe processes that producealcohols, particularly ethanol, by anaerobic fermentation of gasescontaining carbon monoxide. Acetate produced as a by-product of thefermentation process described in WO2007/117157 is converted intohydrogen gas and carbon dioxide gas, either or both of which may be usedin the anaerobic fermentation process.

The fermentation of gaseous substrates comprising CO, to produceproducts such as acids and alcohols, typically favours acid production.Alcohol productivity can be enhanced by methods known in the art, suchas methods described in WO2007/117157, WO2008/115080, WO2009/022925 andWO2009/064200, which are fully incorporated herein by reference.

In order to sustain viability of one or more carboxydotrophic bacteria,such as acetogenic bacteria, a substantially continuous substrate streamcomprising sufficient quantities of CO must be made available to themicrobial culture. Accordingly, if a sufficient amount of CO (or CO2/H2)is not made available to the microbial culture, the culture maydeteriorate and ultimately die. For example during times of insufficientCO supply, such as periods of storage, limited substrate supply orculture/inoculum transfer, a microbial culture will rapidly deplete theavailable CO and viability will deteriorate.

WO2009/114127 provides a method of sustaining viability ofmicroorganisms during periods of limited substrate supply. However, themethod includes adding CO2 to the bioreactor wherein a significantamount of ethanol is converted into acetate, resulting in a decrease inpH. This effect needs to be counteracted to prevent inhibition by excessmolecular acetic acid.

It is an object of the present invention to provide a process that goesat least some way towards overcoming the above disadvantages, or atleast to provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a particular aspect of the invention, there is provided a method ofsustaining viability of a microbial culture of carboxydotrophicbacteria, wherein a substrate comprising CO is limited or unavailable,the method comprising the step of maintaining the culture at atemperature or within a temperature range below the optimum operatingtemperature for growth and/or product production of the microbialculture.

In particular embodiments, the microbial culture is suspended in aliquid nutrient medium.

A substrate is considered to be limited when there is insufficient COavailable to sustain growth and/or metabolite production by themicrobial culture. For example in a continuous culture, the substrate isconsidered to be limited when steady state growth cannot be sustained.

Typically, the substrate comprising CO is consumed by a microbialculture at a rate of at least at least 0.1 mmol/g microbialcells/minute; or at least 0.2 mmol/g/minute; or at least 0.3mmol/g/minute; or at least 0.4 mmol/g/minute; or at least 0.5mmol/g/minute. As such, in particular embodiments, the substratecomprising CO is limited if less than at least 0.1 mmol/g microbialcells/minute; or at least 0.2 mmol/g/minute; or at least 0.3mmol/g/minute; or at least 0.4 mmol/g/minute; or at least 0.5mmol/g/minute is available to the microbial culture. Limitation of thesubstrate is typically associated with a cessation or slowing of growthof the micro-organism.

In certain embodiments of the invention, the temperature of themicrobial culture is reduced to at least 5°; or at least 10°; or atleast 15°; or at least 20°; or at least 25°; or at least 30° below theoptimum operating temperature of the microbial culture. Those skilled inthe art will appreciate upon consideration of the instant disclosure theoptimum operating temperature of a carboxydotrophic bacteria. However,by way of example, Clostridium autoethanogenum has an optimum operatingtemperature of 37° C. As such, in particular embodiments of theinvention, the temperature of the microbial culture is reduced to lessthan 32° C., or less than 30° C., or less than 25° C., or less than 20°C., or less than 15° C., or less than 10° C., or less than 5° C.

In particular embodiments of the invention, the temperature of themicrobial culture can be reduced by cooling the liquid nutrient mediumdirectly or indirectly. In particular embodiments, at least a portion ofthe liquid nutrient medium may be passed through a heat exchanging meansto cool the liquid. Additionally, or alternatively, the microbialculture is contained within a vessel such as a bioreactor or a transportvessel, and the vessel can be cooled by any known cooling means, such asa cooling jacket.

In particular embodiments, the viability of the microbial culture can besubstantially maintained at reduced temperature for at least 3 h, or atleast 5 h, or at least 7 h, or at least 15 h, or at least 30 h, or atleast 48 h.

In another aspect of the invention, there is provided a method ofstoring a microbial culture of a carboxydotrophic bacteria, wherein asubstrate stream is limited or unavailable, the method comprising thestep of reducing the temperature of the microbial culture below theoptimum operating temperature.

In particular embodiments, following storage, for example when asubstrate stream comprising sufficient CO is restored, the temperatureof the microbial culture is increased to the optimum operatingtemperature. In such embodiments, the viability of the microbial cultureis substantially sustained throughout cooling, storage and warming.

In another aspect, there is provided a method of sustaining viability ofa microbial culture during storage, the method including the steps of:

-   -   cooling the microbial culture to a temperature or temperature        range below the optimum operating temperature,    -   storing the microbial culture for a period of time,        In particular embodiments, the method includes warming the        culture to the optimum operating temperature following storage.

In particular embodiments, the extended period is at least 3 h, or atleast 5 h, or at least 7 h, or at least 15 h, or at least 30 h, or atleast 48 h.

In particular embodiments, the method is used to sustain viability of aculture during periods of limited CO supply. In another embodiment, themethod can be used to sustain viability of a microbial culture duringtransport to a remote location. In such embodiments, it is consideredthere may be an insufficient CO supply and/or inadequate agitation tosustain viability. As such, cooling the microbial culture sustainsviability for an extended period.

In particular embodiments of the preceding aspects, storage of theculture includes embodiments wherein the culture is maintained in abioreactor under limited substrate conditions. Additionally oralternatively, the culture can be transferred from a bioreactor to astorage vessel and/or transport vessel. It is expected that in suchembodiments, the culture can be returned to a bioreactor at a latertime.

In particular embodiments, the microbial culture can be used toinoculate a bioreactor following storage. In such embodiments, themicrobial culture may be warmed to the optimum operating temperatureprior to, during or after inoculation.

In another aspect of the invention, there is provided a method oftransporting an inoculum comprising a microbial culture ofcarboxydotrophic bacteria, the method including:

-   -   cooling the microbial culture to below the optimum operating        temperature,    -   transporting the microbial culture to a remote location,    -   inoculating a bioreactor with the microbial culture.

In particular embodiments, the microbial culture is transported to aremote location in a transport vessel. In particular embodiments, themicrobial culture can be cooled and/or warmed in the transport vessel.

In particular embodiments, the method included pressurising thetransport vessel with a substrate comprising CO. In particularembodiments, the transport vessel includes mixing means.

Embodiments of the invention find particular application in theproduction of acids and alcohols, such as ethanol by fermentation of agaseous substrate comprising CO. The substrate may comprise a gasobtained as a by-product of an industrial process. In certainembodiments, the industrial process is selected from the groupconsisting of ferrous metal products manufacturing, non-ferrous productsmanufacturing, petroleum refining processes, gasification of biomass,gasification of coal, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing. In one embodiment of the invention, the gaseous substrateis syngas. In one embodiment, the gaseous substrate comprises a gasobtained from a steel mill.

The CO-containing substrate will typically contain a major proportion ofCO, such as at least about 20% to about 100% CO by volume, from 40% to95% CO by volume, from 40% to 60% CO by volume, and from 45% to 55% COby volume. In particular embodiments, the substrate comprises about 25%,or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO,or about 55% CO, or about 60% CO by volume. Substrates having lowerconcentrations of CO, such as 6%, may also be appropriate, particularlywhen H₂ and CO₂ are also present.

In various embodiments, the fermentation is carried out using a cultureof one or more strains of carboxydotrophic bacteria. In variousembodiments, the carboxydotrophic bacterium is selected fromClostridium, Moorella, Oxobacter, Peptostreptococcus, Acetobacterium,Eubacterium or Butyribacterium. In one embodiment, the carboxydotrophicbacterium is Clostridium autoethanogenum.

The methods of the invention can be used to produce any of a variety ofalcohols, including without limitation ethanol and/or butanol, byanaerobic fermentation of acids in the presence of substrates,particularly gaseous substrates containing carbon monoxide. The methodsof the invention can also be applied to aerobic fermentations, toanaerobic or aerobic fermentations of other products, including but notlimited to isopropanol, and to fermentation of substrates other thancarbon containing gases.

In another aspect, there is provided a system for fermentation of asubstrate comprising CO, including at least one bioreactor; determiningmeans adapted to determine whether the substrate comprising CO isprovided to a microbial culture is limited or non-limited; andtemperature control means configured such that, in use, the temperatureof the bioreactor can be adjusted in response to determination ofwhether the supply of the substrate comprising CO to the microbialculture is limited or non-limited.

In particular embodiments, the controlling means are configured toreduce the temperature of the bioreactor if the determining meansdetermines the supply of the substrate comprising CO is limited. Inparticular embodiments, the system includes processing means configuredsuch that the temperature of the bioreactor can be automaticallyregulated in response to changes in whether the substrate comprising COis limited or non-limited.

In another embodiment, the temperature control mean is configured suchthat the temperature of the bioreactor can be maintained at or about theoptimum operating temperature if the substrate is not limiting.

The invention may also includes the parts, elements and featuresreferred to or indicated in the specification of the application,individually or collectively, in any or all combinations of two or moreof said parts, elements or features, and where specific integers arementioned herein which have known equivalents in the art to which theinvention relates, such known equivalents are deemed to be incorporatedherein as if individually set forth.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the methods of the invention, it has beensurprisingly recognised that carboxydotrophic microbial cultures may bestored with minimal or no additional substrate feeding and/or agitation,at temperatures below their optimum. Accordingly, in particularembodiments, the microbial culture can be transported from one locationto a remote location at temperatures substantially below their growthand/or metabolite production optimum temperature, and may besubsequently used to inoculate a bioreactor. Typically, when acarboxydotrophic microbial culture is stored without providingadditional substrate and/or agitation, the microbial culture willrapidly deplete any CO dissolved in a liquid nutrient medium andviability of the culture deteriorates over time. Consequently, when suchcultures are used to inoculate a bioreactor following storage withoutagitation, there may be a lag time before microbial growth and/orexpected productivity is observed and/or the inoculation may beunsuccessful.

Additionally or alternatively, in particular embodiments, where asubstrate comprising CO, for example a gaseous substrate stream, is notcontinuously available, the microbial culture can be cooled to atemperature below the optimum operating temperature and stored untilfurther substrate is available. In other embodiments where the substrateis limited (i.e. where CO is available but not enough to promote optimumgrowth and/or metabolite production), the microbial culture may becooled to reduce the requirement for CO.

In accordance with particular embodiments of the invention, the methodcan be used to sustain viability of a microbial culture through periodsof limited substrate supply. For example, continuous steady statefermentation of a substrate comprising CO typically requires thesubstrate to be provided in a non-limited manner such that asubstantially constant growth and metabolite production rate issustained. However, the methods of the invention can be used to sustainthe viability of the culture during periods of limited substrate supplywhich would otherwise result in culture deterioration.

Without wishing to be bound by theory, in order to sustain viability ofcarboxydotrophic bacteria such as Clostridium autoethanogenum, CO needsto be supplied to the culture at a rate greater than or equal to the COuptake rate of the microbial culture. For example, under optimumconditions required to promote growth and/or metabolite production, theCO uptake rate of the microbial culture is at least 0.1 mmol/g microbialcells/minute; or at least 0.2 mmol/g/minute; or at least 0.3mmol/g/minute; or at least 0.4 mmol/g/minute; or at least 0.5mmol/g/minute. Accordingly, in particular embodiments where a microbialculture is suspended in a liquid nutrient medium, the culture willrapidly deplete CO dissolved in the medium unless the dissolved CO canbe replenished at a rate equal to or faster than the CO uptake rate.Since CO is poorly soluble in aqueous nutrient media, an external force,such as agitation and/or elevated pressure, is typically required inaddition to a constant supply of a substrate comprising CO to maintaindesirable CO transfer rate into solution. In bioreactors, this istypically achieved by sparging CO into the liquid nutrient medium andoptionally further agitating the liquid to increase the rate of COtransfer into the liquid. Such methods are not generally available invessels suitable for storage of a microbial culture in a liquid nutrientmedium, such as a transport vessel.

It is recognised that in particular embodiments, wherein the microbialculture is being transported from one location to a remote location,there may be a small degree of agitation through movement of the vessel.However, it is considered that the minor agitation associated withvessel transport (i.e. transport by road) is substantially less thanwhat is required to maintain a CO transfer rate into the liquid nutrientmedium to prevent culture deterioration.

In accordance with the methods of the invention, cooling the microbialculture substantially sustains the viability of the culture over anextended period. In particular embodiments, the depletion of CO in astorage vessel can be minimised by cooling the vessel. Additionally oralternatively, the microbial culture may be allowed to cool towards alower ambient temperature. Accordingly, when such cultures areoptionally returned to an optimum temperature (or an optimum temperaturerange) and used to inoculate a bioreactor following storage, microbialgrowth and/or desired productivity is observed more quickly. Suchmethods ameliorate or at least reduce the need for additional CO,sparging and/or agitation.

DEFINITIONS

Unless otherwise defined, the following terms as used throughout thisspecification are defined as follows:

The term “substrate comprising carbon monoxide” and like terms should beunderstood to include any substrate in which carbon monoxide isavailable to one or more strains of bacteria for growth and/orfermentation, for example.

“Gaseous substrate comprising carbon monoxide” include any gas whichcontains carbon monoxide. The gaseous substrate will typically contain asignificant proportion of CO, preferably at least about 5% to about 100%CO by volume.

In the context of fermentation products, the term “acid” as used hereinincludes both carboxylic acids and the associated carboxylate anion,such as the mixture of free acetic acid and acetate present in afermentation broth as described herein. The ratio of molecular acid tocarboxylate in the fermentation broth is dependent upon the pH of thesystem. The term “acetate” includes both acetate salt alone and amixture of molecular or free acetic acid and acetate salt, such as themixture of acetate salt and free acetic acid present in a fermentationbroth as may be described herein. The ratio of molecular acetic acid toacetate in the fermentation broth is dependent upon the pH of thesystem.

The term “bioreactor” includes a fermentation device consisting of oneor more vessels and/or towers or piping arrangements, which includes theContinuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR),Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, MembraneReactor such as Hollow Fibre Membrane Bioreactor (HFMBR), Static Mixer,or other vessel or other device suitable for gas-liquid contact.

Unless the context requires otherwise, the phrases “fermenting”,“fermentation process” or “fermentation reaction” and the like, as usedherein, are intended to encompass both the growth phase and productbiosynthesis phase of the process. As will be described further herein,in some embodiments the bioreactor may comprise a first growth reactorand a second fermentation reactor. As such, the addition of metals orcompositions to a fermentation reaction should be understood to includeaddition to either or both of these reactors.

Unless the context requires otherwise, the phrases “storage” and “store”are used in reference to periods when a microbial culture has a limitedsubstrate supply or a substrate is unavailable. As such, the termincludes periods when a microbial culture under steady state growthconditions is temporarily unavailable limited in substrate supply andincludes periods when a microbial culture is transferred from abioreactor into a storage vessel, such as an inoculum transfer vessel.

The term “overall net conversion” and the like, as used herein, isintended to describe the conversion of substrates, such as CO, toproducts including acid(s) and/or alcohol(s) by a microbial culture at aparticular time point. It is recognised that portions of a microbialculture may be devoted to different functions at a particular time pointand a number of products may be produced. Furthermore, one or more ofthe products present in the fermentation broth may be converted intoother products. Accordingly, the overall net conversion includes all theproducts produced by the microbial culture at any particular point intime.

While the following description focuses on particular embodiments of theinvention, namely the production of ethanol and/or acetate using CO asthe primary substrate, it should be appreciated that the invention maybe applicable to production of alternative alcohols and/or acids and theuse of alternative substrates as will be known by persons of ordinaryskill in the art to which the invention relates. For example, gaseoussubstrates containing carbon dioxide and hydrogen may be used. Further,the invention may be applicable to fermentation to produce butyrate,propionate, caproate, ethanol, propanol, and butanol. The methods mayalso be of use in producing hydrogen. By way of example, these productsmay be produced by fermentation using microbes from the genus Moorella,Clostridia, Ruminococcus, Acetobacterium, Eubacterium, Butyribacterium,Oxobacter, Methanosarcina, and Desulfotomaculum.

Certain embodiments of the invention are adapted to use gas streamsproduced by one or more industrial processes. Such processes includesteel making processes, particularly processes which produce a gasstream having a high CO content or a CO content above a predeterminedlevel (i.e., 5%). According to such embodiments, acetogenic bacteria arepreferably used to produce acids and/or alcohols, particularly ethanolor butanol, within one or more bioreactors. Those skilled in the artwill be aware upon consideration of the instant disclosure that theinvention may be applied to various industries or waste gas streams,including those of vehicles with an internal combustion engine. Also,those skilled in the art will be aware upon consideration of the instantdisclosure that the invention may be applied to other fermentationreactions including those using the same or different micro-organisms.It is therefore intended that the scope of the invention is not limitedto the particular embodiments and/or applications described but isinstead to be understood in a broader sense; for example, the source ofthe gas stream is not limiting, other than that at least a componentthereof is usable to feed a fermentation reaction. The invention hasparticular applicability to improving the overall carbon capture and/orproduction of ethanol and other alcohols from gaseous substrates such asautomobile exhaust gases and high volume CO-containing industrial fluegases.

Fermentation

Processes for the production of ethanol and other alcohols from gaseoussubstrates are known. Exemplary processes include those described forexample in WO2007/117157, WO2008/115080, U.S. Pat. No. 6,340,581, U.S.Pat. No. 6,136,577, U.S. Pat. No. 5,593,886, U.S. Pat. No. 5,807,722 andU.S. Pat. No. 5,821,111, each of which is incorporated herein byreference.

A number of anaerobic bacteria are known to be capable of carrying outthe fermentation of CO to alcohols, including n-butanol and ethanol, andacetic acid, and are suitable for use in the process of the presentinvention. Examples of such bacteria that are suitable for use in theinvention include those of the genus Clostridium, such as strains ofClostridium Ijungdahlii, including those described in WO 00/68407, EP117309, U.S. Pat. Nos. 5,173,429, 5,593,886, and 6,368,819, WO 98/00558and WO 02/08438, Clostridium carboxydivorans (Liou et al., InternationalJournal of Systematic and Evolutionary Microbiology 33: pp 2085-2091),Clostridium ragsdalei (WO/2008/028055) and Clostridium autoethanogenum(Abrini et al, Archives of Microbiology 161: pp 345-351). Other suitablebacteria include those of the genus Moorella, including Moorella spHUC22-1, (Sakai et al, Biotechnology Letters 29: pp 1607-1612), andthose of the genus Carboxydothermus (Svetlichny, V. A., Sokolova, T. G.et al (1991), Systematic and Applied Microbiology 14: 254-260). Furtherexamples include Moorella thermoacetica, Moorella thermoautotrophica,Ruminococcus productus, Acetobacterium woodii, Eubacterium limosum,Butyribacterium methylotrophcum, Oxobacter pfennigii, Methanosarcinabarkeri, Methanosarcina acetivorans, Desulfotomaculum kuznetsovii (Simpaet. al. Critical Reviews in Biotechnology, 2006 Vol. 26. Pp 41-65). Inaddition, it should be understood that other acetogenic anaerobicbacteria may be applicable to the present invention as would beunderstood by a person of skill in the art. It will also be appreciatedthat the invention may be applied to a mixed culture of two or morebacteria.

One exemplary micro-organism suitable for use in the present inventionis Clostridium autoethanogenum. In one embodiment, the Clostridiumautoethanogenum is a Clostridium autoethanogenum having the identifyingcharacteristics of the strain deposited at the German Resource Centrefor Biological Material (DSMZ) under the identifying deposit number19630. In another embodiment, the Clostridium autoethanogenum is aClostridium autoethanogenum having the identifying characteristics ofDSMZ deposit number DSMZ 10061.

Culturing of the bacteria used in the methods of the invention may beconducted using any number of processes known in the art for culturingand fermenting substrates using anaerobic bacteria. Exemplary techniquesare provided in the “Examples” section below. By way of further example,those processes generally described in the following articles usinggaseous substrates for fermentation may be utilised: (i) K. T. Klasson,et al. (1991). Bioreactors for synthesis gas fermentations resources.Conservation and Recycling, 5; 145-165; (ii) K. T. Klasson, et al.(1991). Bioreactor design for synthesis gas fermentations. Fuel. 70.605-614; (iii) K. T. Klasson, et al. (1992). Bioconversion of synthesisgas into liquid or gaseous fuels. Enzyme and Microbial Technology. 14;602-608; (iv) J. L. Vega, et al. (1989). Study of Gaseous SubstrateFermentation Carbon Monoxide Conversion to Acetate. 2. ContinuousCulture. Biotech. Bioeng. 34. 6. 785-793; (v) J. L. Vega, et al. (1989).Study of gaseous substrate fermentations: Carbon monoxide conversion toacetate. 1. Batch culture. Biotechnology and Bioengineering. 34. 6.774-784; (vi) J. L. Vega, et al. (1990). Design of Bioreactors for CoalSynthesis Gas Fermentations. Resources, Conservation and Recycling. 3.149-160; all of which are incorporated herein by reference.

The fermentation may be carried out in any suitable bioreactor, such asa continuous stirred tank reactor (CSTR), an immobilised cell reactor, agas-lift reactor, a bubble column reactor (BCR), a membrane reactor,such as a Hollow Fibre Membrane Bioreactor (HFMBR) or a trickle bedreactor (TBR). Also, in some embodiments of the invention, thebioreactor may comprise a first, growth reactor in which themicro-organisms are cultured, and a second, fermentation reactor, towhich fermentation broth from the growth reactor is fed and in whichmost of the fermentation product (e.g. ethanol and acetate) is produced.

According to various embodiments of the invention, the carbon source forthe fermentation reaction is a gaseous substrate containing CO. Thesubstrate may be a CO-containing waste gas obtained as a by-product ofan industrial process, or from some another source such as fromautomobile exhaust fumes. In certain embodiments, the industrial processis selected from the group consisting of ferrous metal productsmanufacturing, such as a steel mill, non-ferrous products manufacturing,petroleum refining processes, gasification of coal, electric powerproduction, carbon black production, ammonia production, methanolproduction and coke manufacturing. In these embodiments, theCO-containing substrate may be captured from the industrial processbefore it is emitted into the atmosphere, using any convenient method.Depending on the composition of the CO-containing substrate, it may alsobe desirable to treat it to remove any undesired impurities, such asdust particles before introducing it to the fermentation. For example,the gaseous substrate may be filtered or scrubbed using known methods.

Alternatively, the CO-containing substrate may be sourced from thegasification of biomass. The process of gasification involves partialcombustion of biomass in a restricted supply of air or oxygen. Theresultant gas typically comprises mainly CO and H₂, with minimal volumesof CO₂, methane, ethylene and ethane. For example, biomass by-productsobtained during the extraction and processing of foodstuffs such assugar from sugarcane, or starch from maize or grains, or non-foodbiomass waste generated by the forestry industry may be gasified toproduce a CO-containing gas suitable for use in the present invention.

The CO-containing substrate will typically contain a major proportion ofCO, such as at least about 20% to about 100% CO by volume, from 40% to95% CO by volume, from 60% to 90% CO by volume, and from 70% to 90% COby volume. In particular embodiments, the substrate comprises 25%, or30%, or 35%, or 40%, or 45%, or 50% CO by volume. Substrates havinglower concentrations of CO, such as 6%, may also be appropriate,particularly when H₂ and CO₂ are also present.

While it is not necessary for the substrate to contain any hydrogen, thepresence of H₂ should not be detrimental to product formation inaccordance with methods of the invention. In particular embodiments, thepresence of hydrogen results in an improved overall efficiency ofalcohol production. For example, in particular embodiments, thesubstrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H2:CO. Inother embodiments, the substrate stream comprises low concentrations ofH2, for example, less than 5%, or less than 4%, or less than 3%, or lessthan 2%, or less than 1%, or is substantially hydrogen free. Thesubstrate may also contain some CO₂ for example, such as about 1% toabout 80% CO₂ by volume, or 1% to about 30% CO₂ by volume.

Typically, the carbon monoxide will be added to the fermentationreaction in a gaseous state. However, the methods of the invention arenot limited to addition of the substrate in this state. For example, thecarbon monoxide can be provided in a liquid. For example, a liquid maybe saturated with a carbon monoxide containing gas and that liquid addedto the bioreactor. This may be achieved using standard methodology. Byway of example a microbubble dispersion generator (Hensirisak et. al.Scale-up of microbubble dispersion generator for aerobic fermentation;Applied Biochemistry and Biotechnology Volume 101, Number 3/October,2002) could be used for this purpose.

It will be appreciated that for growth of the bacteria and CO-to-alcoholfermentation to occur, in addition to the CO-containing substrate gas, asuitable liquid nutrient medium will need to be fed to the bioreactor. Anutrient medium will contain vitamins and minerals sufficient to permitgrowth of the micro-organism used. Anaerobic media suitable for thefermentation of ethanol using CO as the sole carbon source are known inthe art. For example, suitable media are described in U.S. Pat. Nos.5,173,429 and 5,593,886 and WO 02/08438, WO2007/115157 and WO2008/115080referred to above. The present invention provides a novel media whichhas increased efficacy in supporting growth of the micro-organismsand/or alcohol production in the fermentation process. This media willbe described in more detail hereinafter.

The fermentation should desirably be carried out under appropriateconditions for the desired fermentation to occur (e.g. CO-to-ethanol).Reaction conditions that should be considered include pressure,temperature, gas flow rate, liquid flow rate, media pH, media redoxpotential, agitation rate (if using a continuous stirred tank reactor),inoculum level, maximum gas substrate concentrations to ensure that COin the liquid phase does not become limiting, and maximum productconcentrations to avoid product inhibition. Suitable conditions aredescribed in WO02/08438, WO07/117,157 and WO08/115,080.

The optimum reaction conditions will depend partly on the particularmicro-organism used. However, in general, it is preferred that thefermentation be performed at pressure higher than ambient pressure.Operating at increased pressures allows a significant increase in therate of CO transfer from the gas phase to the liquid phase where it canbe taken up by the micro-organism as a carbon source for the productionof ethanol. This in turn means that the retention time (defined as theliquid volume in the bioreactor divided by the input gas flow rate) canbe reduced when bioreactors are maintained at elevated pressure ratherthan atmospheric pressure.

Also, since a given CO-to-ethanol conversion rate is in part a functionof the substrate retention time, and achieving a desired retention timein turn dictates the required volume of a bioreactor, the use ofpressurized systems can greatly reduce the volume of the bioreactorrequired, and consequently the capital cost of the fermentationequipment. According to examples given in U.S. Pat. No. 5,593,886,reactor volume can be reduced in linear proportion to increases inreactor operating pressure, i.e. bioreactors operated at 10 atmospheresof pressure need only be one tenth the volume of those operated at 1atmosphere of pressure.

The benefits of conducting a gas-to-ethanol fermentation at elevatedpressures have also been described elsewhere. For example, WO 02/08438describes gas-to-ethanol fermentations performed under pressures of 30psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369g/l/day respectively. However, example fermentations performed usingsimilar media and input gas compositions at atmospheric pressure werefound to produce between 10 and 20 times less ethanol per litre per day.

It is also desirable that the rate of introduction of the CO-containinggaseous substrate is such as to ensure that the concentration of CO inthe liquid phase does not become limiting. This is because a consequenceof CO-limited conditions may be that the ethanol product is consumed bythe culture.

Product Recovery

The products of the fermentation reaction can be recovered using knownmethods. Exemplary methods include those described in WO07/117,157,WO08/115,080, U.S. Pat. No. 6,340,581, U.S. Pat. No. 6,136,577, U.S.Pat. No. 5,593,886, U.S. Pat. No. 5,807,722 and U.S. Pat. No. 5,821,111.However, briefly and by way of example only ethanol may be recoveredfrom the fermentation broth by methods such as fractional distillationor evaporation, and extractive fermentation.

Distillation of ethanol from a fermentation broth yields an azeotropicmixture of ethanol and water (i.e., 95% ethanol and 5% water). Anhydrousethanol can subsequently be obtained through the use of molecular sieveethanol dehydration technology, which is also well known in the art.

Extractive fermentation procedures involve the use of a water-misciblesolvent that presents a low toxicity risk to the fermentation organism,to recover the ethanol from the dilute fermentation broth. For example,oleyl alcohol is a solvent that may be used in this type of extractionprocess. Oleyl alcohol is continuously introduced into a fermenter,whereupon this solvent rises forming a layer at the top of the fermenterwhich is continuously extracted and fed through a centrifuge. Water andcells are then readily separated from the oleyl alcohol and returned tothe fermenter while the ethanol-laden solvent is fed into a flashvaporization unit. Most of the ethanol is vaporized and condensed whilethe oleyl alcohol is non volatile and is recovered for re-use in thefermentation.

Acetate, which is produced as a by-product in the fermentation reaction,may also be recovered from the fermentation broth using methods known inthe art.

For example, an adsorption system involving an activated charcoal filtermay be used. In this case, it is preferred that microbial cells arefirst removed from the fermentation broth using a suitable separationunit. Numerous filtration-based methods of generating a cell freefermentation broth for product recovery are known in the art. The cellfree ethanol—and acetate—containing permeate is then passed through acolumn containing activated charcoal to adsorb the acetate. Acetate inthe add form (acetic acid) rather than the salt (acetate) form is morereadily adsorbed by activated charcoal. It is therefore preferred thatthe pH of the fermentation broth is reduced to less than about 3 beforeit is passed through the activated charcoal column, to convert themajority of the acetate to the acetic acid form.

Acetic acid adsorbed to the activated charcoal may be recovered byelution using methods known in the art. For example, ethanol may be usedto elute the bound acetate. In certain embodiments, ethanol produced bythe fermentation process itself may be used to elute the acetate.Because the boiling point of ethanol is 78.8° C. and that of acetic acidis 107° C., ethanol and acetate can readily be separated from each otherusing a volatility-based method such as distillation.

Other methods for recovering acetate from a fermentation broth are alsoknown in the art and may be used in the processes of the presentinvention. For example, U.S. Pat. Nos. 6,368,819 and 6,753,170 describea solvent and cosolvent system that can be used for extraction of aceticacid from fermentation broths. As with the example of the oleylalcohol-based system described for the extractive fermentation ofethanol, the systems described in U.S. Pat. Nos. 6,368,819 and 6,753,170describe a water immiscible solvent/co-solvent that can be mixed withthe fermentation broth in either the presence or absence of thefermented micro-organisms in order to extract the acetic acid product.The solvent/co-solvent containing the acetic acid product is thenseparated from the broth by distillation. A second distillation step maythen be used to purify the acetic acid from the solvent/co-solventsystem.

The products of the fermentation reaction (for example ethanol andacetate) may be recovered from the fermentation broth by continuouslyremoving a portion of the broth from the fermentation bioreactor,separating microbial cells from the broth (conveniently by filtration),and recovering one or more product from the broth simultaneously orsequentially. In the case of ethanol it may be conveniently recovered bydistillation, and acetate may be recovered by adsorption on activatedcharcoal, using the methods described above. The separated microbialcells are preferably returned to the fermentation bioreactor. The cellfree permeate remaining after the ethanol and acetate have been removedis also preferably returned to the fermentation bioreactor. Additionalnutrients (such as B vitamins) may be added to the cell free permeate toreplenish the nutrient medium before it is returned to the bioreactor.Also, if the pH of the broth was adjusted as described above to enhanceadsorption of acetic acid to the activated charcoal, the pH should bere-adjusted to a similar pH to that of the broth in the fermentationbioreactor, before being returned to the bioreactor.

Sustaining Culture Viability

In accordance with the invention, there is provided, a method ofsubstantially sustaining viability of a microbial culture ofcarboxydotrophic bacteria, wherein substrate comprising CO is limited orunavailable, the method comprising reducing the temperature of themicrobial culture below the optimum temperature for growth and/orproduct production.

In accordance with the methods of the invention, culture viability issustained if, on warming to optimum temperature (or range), the culturecan resume metabolism to produce products and/or cell growth. Inparticular embodiments, the microbial culture can be used to inoculate abioreactor and sustaining culture viability ensures the culture canresume metabolism following transfer. It is recognised that a microbialculture stored in accordance with the methods of the invention maycontinue to metabolise and/or grow albeit at a slower rate. However, onrestoring the temperature of the microbial culture toward the optimum,metabolism and/or growth rate are expected to increase to pre-storagelevels.

A microbial culture becomes limited in CO when the rate of transfer ofCO into an aqueous nutrient medium is slower than the rate at which themicrobial culture can take up (or consume) the CO. Typically,carboxydotrophic bacteria, such as Clostridium autoethanogenum, uptakeCO from a liquid nutrient medium at a rate greater than 0.1 mmol/gmicrobial cells/minute; or at least 0.2 mmol/g/minute; or at least 0.3mmol/g/minute; or at least 0.4 mmol/g/minute; or at least 0.5mmol/g/minute. Accordingly, it is generally necessary to provide aconstant stream of a substrate comprising CO to the microbial culture.Furthermore, due to the low solubility of CO in aqueous systems, it istypically necessary to further increase the CO transfer rates (masstransfer), for example by increasing partial pressure of CO in thesubstrate stream and/or agitation of the liquid nutrient medium. Uponconsideration of the instant disclosure, those skilled in the art willappreciate alternative methods of increasing mass transfer of CO inaccordance with particular embodiments of the invention.

In particular embodiments, the methods of the invention can be used tosustain the viability of a microbial culture, wherein the microbialculture is limited in CO, such that the rate of transfer of CO intosolution is less than the uptake rate of the culture. Such situationsmay arise when a substrate comprising CO is not continuously provided tothe microbial culture; the mass transfer rate is low; or there isinsufficient CO in a substrate stream to sustain culture vitality atoptimum temperature. In such embodiments, the microbial culture willrapidly deplete the CO dissolved in the liquid nutrient medium andbecome substrate limited as further substrate cannot be provided fastenough. Unless the microbial culture is cooled in accordance with themethods of the invention, the viability of the microbial culture willdiminish over time, resulting in complete culture death, or the culturedeteriorating to such a level where it is no longer limited by theconditions.

For example, in particular embodiments, a microbial culture comprisingone or more carboxydotrophic micro-organisms can be operated undersubstantially steady state conditions when a substrate comprising CO isnot limited. Under such conditions, it is expected the microbial culturewill have a substantially constant growth rate and substantiallyconstant metabolite(s) production rate. However, when the substratecannot be provided in a non-limited way, microbial growth will slow orcease and the microbial culture will rapidly deteriorate and will washout of a continuously purged bioreactor. Under such conditions, even ifthe substrate is returned to a non-limited supply, the culture may notbe revived, or at least revival takes an extended period. However, inaccordance with the invention, the temperature of the culture isdecreased, such that viability of the microbial culture is sustainedduring storage periods of limited or no substrate supply.

It is recognised that the metabolism of the microbial culture may slowwhen the temperature is decreased, so operating conditions, such as cellretention times may need to be adjusted.

In particular embodiments of the invention, the stored microbial cultureis used for inoculation of a bioreactor. In such embodiments, it isdesirable that the culture is suitably dense (i.e. large number ofmicrobes per unit volume) and that the viability of the culture issubstantially sustained during storage (i.e. transport to a remotelocation). Typically, the higher the density of the microbial cells inthe culture, the faster they will deplete any CO available in a liquidnutrient medium. Without wishing to be bound by theory, it is consideredthat when CO is not available, or is sufficiently depleted, theviability of the microbial culture decreases. For example, at least aportion of the culture begins to die off and/or the culture switches toa slower metabolism, such that when a bioreactor is inoculated with themicrobial culture, there is a lag before high growth rates and/orproductivity is attained. However, when the culture is cooled, thedepletion of CO in the liquid nutrient medium is slowed such that theculture viability is substantially preserved over an extended period.

In accordance with the methods of the invention, the culture may becooled to a temperature below the optimum growth and/or metaboliteproduction temperature, such that viability of the culture is sustainedover an extended period. Typically, carboxydotrophic micro-organismshave an optimum operating temperatures of carboxydotrophic bacteria inthe range 30-70° C. Examples of optimum operating temperature aredetailed in “Microbiology of synthesis gas fermentation for biofuelsproduction” A. M. Henstra et al. Current Opinion in Biotechnology, 2007,18, 200-206. For example, mesophilic bacteria, such as Clostridiumautoethanogenum, Clostridium Ijungdahli and Clostridium carboxydivoranshave an optimum growth and metabolite production temperature ofapproximately 37° C. However, thermophilic bacteria have significantlyhigher optimum temperatures of 55-70° C., for example strains ofMoorella thermoacetica (55-60° C.), Carboxydothermus hydrogenoformans(70-72° C.), Desulfotomaculum carboxydivorans (60° C.). As such, inaccordance with the methods of the invention, it is necessary to coolthe microbial culture to at least 2°; or at least 5°; or at least 10°;at least 15°; or at least 20°; or at least 25°; or at least 30° belowthe optimum temperature to sustain culture viability. For example,Clostridium autoethanogenum can be cooled to less than 30° C., or lessthan 25° C., or less than 20° C., or less than 15° C., or less than 10°C., or less than 5° C.

In accordance with the methods of the invention, on cooling, viabilityof the culture is sustained for extended periods, even in the absence ofadditional substrate comprising CO and/or agitation. In particularembodiments, viability of the culture is sustained for at least 3 h, orat least 5 h, or at least 7 h, or at least 15 h, or at least 30 h, or atleast 48 h. For example; Clostridium autoethanogenum remains viable forat least 30 hours, when stored at reduced temperature.

Those skilled in the art will appreciate means required to cool amicrobial culture will depend on several factors including size andshape of the vessel containing the culture, speed at which the cultureis cooled and whether the culture is exothermic or endothermic. Forexample, many large scale fermentation processes need to be externallycooled to remove excess heat generated during the fermentation reaction.The known cooling means already provided may be adapted to further coolthe microbial culture to sustain viability. In alternative embodiments,where the microbial culture requires external heating to maintain theoptimum operating temperature, the culture may be cooled by removing theheat source and allowing the fermenter to cool to ambient temperatureover time. Additionally or alternatively, such cultures may be furthercooled using any known refrigeration or cooling means.

In particular embodiments of the invention, the liquid nutrient media isallowed to cool below the optimum operating temperature by removingthermostatic heat control. Under such conditions, the temperature of theliquid nutrient media and the microbial culture will fall toward ambienttemperature over time. In accordance with the invention, as thetemperature of the microbial culture falls below the optimum operatingtemperature, alcohol productivity increases.

It is considered that periods where viability of a microbial culture maybe sustained using the methods of the invention will be commonlyencountered in industrial fermentation processes, as continuity of asubstrate stream comprising CO may not be guaranteed. For example, wherea substrate comprising CO is derived from an industrial process, such asoff-gas from a steel mill, there may be occasions where the industrialprocess (i.e. steel manufacture) is slowed or shut down for extendedperiods. Under such conditions, the production of a substrate comprisingCO will slow or stop altogether. Consequently, when CO supply is limitedor CO is unavailable to a bioreactor containing a carboxydotrophicmicrobial culture, the viability of the culture will diminish over time.However, in accordance with the methods of the invention, if the cultureis cooled, the viability can be sustained during CO limited operation.

Similarly, when syngas produced from the gasification of feedstock'ssuch as biomass or municipal solid waste is used as the substratestream, there may be times when the CO content of the stream decreases,or the gasifier has to be taken off-line, for maintenance (for example).Again, under such conditions, viability of a microbial culture requiringCO for metabolism will deteriorate unless the culture can be cooled inaccordance with the methods of the invention.

In an alternative embodiment, the methods of the invention can be usedto substantially sustain the viability of a microbial culture used forinoculation of a remote bioreactor. For example, a microbial culture canbe placed in a vessel suitable for transport and transported to a remotelocation. Typically, the transport vessel would require a supply of COand agitation means to ensure viability of the culture was sustained,both of which can be difficult to provide in mobile environments.However, in accordance with the methods of the invention, the microbialculture can be cooled in the transport vessel such that viability of theinoculum is sustained during transport, even in the absence of asufficient supply of CO and/or agitation.

In accordance with another embodiment of the invention, there isprovided a system for fermentation of a substrate comprising CO,including at least one bioreactor; determining means adapted todetermine whether the substrate comprising CO is provided to a microbialculture is limited or non-limited; and temperature control meansconfigured such that, in use, the temperature of the bioreactor can beadjusted in response to determination of whether the supply of thesubstrate comprising CO to the microbial culture is limited ornon-limited.

In particular embodiments, wherein the determining means determine thatthe substrate supply has become limited, the temperature of themicrobial culture can be decreased to sustain culture viability.Additionally or alternatively, wherein the determining means determinesthe substrate is not limited, the temperature can be maintainedsubstantially at optimum operating temperature. In particularembodiments, the system includes processing means, such that in use, thecontrolling means can regulate the temperature of the microbial cultureautomatically in accordance with the methods of the invention.

FIG. 1 is a schematic representation of a system 100 according to oneembodiments of the invention. Input substrate stream 1 enters bioreactor2 via a suitable conduit. Input substrate stream 1 comprises CO and inaccordance with the methods of the invention, the rate of supply and/orthe composition of the substrate stream 1 may vary. The system 100includes determining means 3 which, in use, determine whether thesubstrate supplied to a microbial culture in the bioreactor is limited.The system 100 includes temperature control means 4, which can regulatethe temperature of the bioreactor 1 such that a microbial culture can bemaintained at an optimum operating temperature, or the temperaturedecreased and/or maintained at a temperature below the optimum operatingtemperature.

In particular embodiments, the temperature control means 4 is configuredsuch that in use, if the determining means determines the substratesupply is not limited, the temperature of the fermentation can bemaintained at or around the optimum operating temperature. Additionallyor alternatively, if the determining means 3 determines that thesubstrate supply is limited, the controlling means 4 can decrease thetemperature of the bioreactor 1 in accordance with the methods of theinvention. Thus, the temperature can be controlled at a temperaturesubstantially below the optimum operating temperature until thesubstrate supply is no longer limiting.

In particular embodiments, the system 100 includes optional processingmeans 5 configured to regulate the controlling means 4 automatically, inresponse to determinations made by the determining means 3.

EXAMPLES Materials and Methods Preparation of Media LM33:

Concentration per Media Component 1.0 L of Media MgCl₂•6H₂O 0.5 g NaCl0.2 g CaCl₂•6H₂O 0.26 g NaH₂PO₄ 2.04 g KCl 0.15 g NH₄Cl 2.5 g Compositetrace metal solution (LS06) 10 mL Composite B vitamin solution (LS03) 10mL Resazurin (2 g/L stock) 1 mL FeCl₃ (5 g/L stock) 2 mL Cysteine HCl0.5 g Distilled water Up to 1 L

Composite B vitamin Solution (LS03) per L of Stock Biotin 20.0 mg Folicacid 20.0 mg Pyridoxine hydrochloride 10.0 mg Thiamine•HCl 50.0 mgRiboflavin 50.0 mg Nicotinic acid 50.0 mg Calcium D-(*)-pantothenate50.0 mg Vitamin B12 50.0 mg p-Aminobenzoic acid 50.0 mg Thioctic acid50.0 mg Distilled water To 1 Litre

Composite trace metal solution (LSO6) per L of stock NitrilotriaceticAcid 1.5 g MgSO₄•7H₂O 3.0 g MnSO₄•H₂O 0.5 g NaCl 1.0 g FeSO₄•7H₂O 0.1 gFe(SO₄)₂(NH₄)₂•6H₂O 0.8 g CoCl₂•6H₂O 0.2 g ZnSO₄•7H₂O 0.2 g CuCl₂•2H₂O0.02 g AlK(SO₄)₂•12H₂O 0.02 g H₃BO₃ 0.30 g NaMoO₄•2H₂O 0.03 g Na₂SeO₃0.02 g NiCl₂•6H₂O 0.02 g Na₂WO₄•6H₂O 0.02 g

Media was prepared at pH 5.5 as follows. All ingredients with theexception of Cysteine-HCl were mixed in 400 ml distilled water. Thissolution was made anaerobic by heating to boiling and allowing it tocool to room temperature under a constant flow of N2 gas. Once cool, theCysteine-HCl was added and the pH of the solution adjusted to 5.5 beforemaking the volume up to 1000 ml; anaerobicity was maintained throughoutthe experiments.

Bacteria: Clostridium autoethanogenum were obtained from the GermanResource Centre for Biological Material (DSMZ). The accession numbergiven to the bacteria is DSMZ 19630.

Typical Continuous Culture in Bioreactor at Atmospheric Pressure forInoculum

A five-litre bioreactor was filled with 4900 ml of the media LM33without Composite B vitamin solution (LS03) or Cysteine-HCl andautoclaved for 30 minutes at 121° C. While cooling down, the media wassparged with N2 to ensure anaerobicity. Cysteine-HCl and Composite Bvitamin solution (LS03) were then added. Anaerobicity was maintainedthroughout the fermentation. The gas was switched to 95% CO, 5% CO₂ atatmospheric pressure prior to inoculation with 100 ml of a Clostridiumautoethanogenum culture. The bioreactor was maintained at 37° C. stirredat 200 rpm at the start of the culture. During the growth phase, theagitation was increased to 400 rpm. The pH was set to 5.5 and maintainedby automatic addition of 5 M NaOH. Fresh anaerobic media wascontinuously added into the bioreactor to maintain a defined biomass andacetate level the bioreactor.

Example 1

Sterile serum bottles were purged three times with CO containing gas(20% CO2; 30% N2 and 3% H2 in CO) and then evacuated to a vacuum of −5psi. 50 ml of active culture containing biomass, acetate and traces ofethanol under atmospheric pressure was transferred directly from acontinuous bioreactor into the 234 ml serum bottle. The 184 ml headspacewas then filled with the CO containing gas to 40 psia and incubatedwithout shaking at the indicated temperature.

After 3, 6, 24 and 31 hours of incubation, a 2 ml sample from each serumvial was transferred into a new serum vial containing 50 ml of media(LM33) prepared in accordance with the above. The vials were filled withthe CO containing gas to 40 psia and incubated at 37° C. for severaldays with constant agitation.

Growth of the inoculated vials was visually assessed at time intervalsand −/+/++ were assigned to describe no growth, slight growth and densegrowth respectively (see Table 1).

TABLE 1 Growth of inoculated Clostridium autoethanogenum culturefollowing storage at various temperatures over 3, 6, 24 and 31 h.Incubation time 3 h 6 h 24 h 31 h Incubation Days following inoculationtemp 0 1 2 0 1 2 0 1 2 0 1 2  4° C. − + ++ − + ++ − + ++ − + ++ 14° C.− + ++ − + ++ − + ++ − − + 24° C. − + ++ − + + − − − − − − 37° C. − − −− − − − − − − − −

The optimum temperature for production of products and microbial growthof Clostridium autoethanogenum is 37° C. At 37° C., the non shaken vialswere either non-viable or had substantially reduced viability when usedfor inoculation after 3, 6, 24 and 31 hours. It is considered thatwithout agitation, the active microbial culture rapidly depletes thelimited CO dissolved in the liquid nutrient medium. The excess carbonmonoxide in the headspace may have limited transfer into the liquidnutrient medium. However, in the absence of agitation, it is expectedthere will be a CO gradient, wherein the uppermost surface of the liquidnutrient medium may have a relatively high CO concentration, but thiswill decrease down through the medium. In the absence of agitation, themicrobial cells will settle to the bottom of the vial, where they willbe substantially starved of substrate and will rapidly decrease inviability. Subsequently, the deteriorated or dead culture is unsuitablefor inoculation.

On reducing the temperature of the stored culture to 24° C., themicrobial culture remained substantially viable for inoculation of abioreactor for over 3 h. At 14° C., the microbial culture remainedsubstantially viable following storage for 3 h, 6 h and 24 h. Following31 h storage, the microbial culture remained viable, but took longer togrow following inoculation. At 4° C., the microbial culture remainedviable following storage over all times investigated.

The invention has been described herein with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. Those skilled in the art willappreciate that the invention is susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the invention includes all such variations andmodifications. Furthermore, titles, heading, or the like are provided toenhance the reader's comprehension of this document, and should not beread as limiting the scope of the present invention. The entiredisclosures of all applications, patents and publications cited aboveand below, if any, are herein incorporated by reference.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgement or any form of suggestion that thatprior art forms part of the common general knowledge in the field ofendeavour in any country in the world.

Throughout this specification and any claims which follow, unless thecontext requires otherwise, the words “comprise”, “comprising” and thelike, are to be construed in an inclusive sense as opposed to anexclusive sense, that is to say, in the sense of “including, but notlimited to”.

1-24. (canceled)
 25. A method of sustaining viability of a microbialculture of carboxydotrophic bacteria when a substrate that includes COis limited or unavailable, said method comprising maintaining theculture substantially at a temperature or within a temperature rangebelow an optimum operating temperature of the culture.
 26. The method ofclaim 25, wherein maintaining the culture comprises maintaining thetemperature at least 5° C. below the optimum operating temperature. 27.The method of claim 25, further comprising maintaining the temperatureof the microbial culture substantially at the optimum operatingtemperature if the substrate comprising CO becomes non-limited.
 28. Themethod of claim 25, wherein the microbial culture is in liquid nutrientmedia in a bioreactor.
 29. The method of claim 28, further comprisingcooling the bioreactor such that the temperature of the liquid nutrientmedia is maintained at a temperature below the optimum operatingtemperature.
 30. The method of claim 25, further comprising sustainingviability of the culture over a period of limited CO availability of atleast 3 hours.
 31. The method of claim 25, wherein the carboxydotrophicbacteria is selected from the group consisting of Clostridium, Moorella,Pyrococcus, Eubacterium, Desulfobacterium, Carboxydothermus,Acetogenium, Acetobacterium, Acetoanaerobium, Butyribaceterium andPeptostreptococcus.
 32. The method of claim 25, wherein thecarboxydotrophic bacteria is Clostridium autoethanogenum.
 33. The methodof claim 25, wherein the substrate comprises a gas obtained as aby-product of an industrial process selected from the group consistingof ferrous metal products manufacturing, non-ferrous productsmanufacturing, petroleum refining processes, gasification of biomass,gasification of coal, electric power production, carbon blackproduction, ammonia production, methanol production and cokemanufacturing.
 34. The method of claim 25, wherein the substrate thatincludes CO comprises at least about 15% to about 100% CO by volume. 35.A method of sustaining viability of a microbial culture ofcarboxydotrophic bacteria during storage, said method comprising:cooling the microbial culture to a temperature or temperature rangebelow an optimum operating temperature; and storing the microbialculture under limited CO conditions for a selected period of time. 36.The method of claim 35, further comprising returning the culture to theoptimum operating temperature under non-limited CO conditions.
 37. Themethod of claim 35, wherein storing the microbial culture comprisesstoring the microbial culture for at least 5 hours.
 38. The method ofclaim 37, further comprising transporting the microbial culture to aremote location during storage.
 39. The method of claim 37, furthercomprising inoculating the bioreactor with the microbial culturefollowing storage.
 40. The method of claim 35, wherein thecarboxydotrophic bacteria are selected from the group consisting ofClostridium, Moorella, Pyrococcus, Eubacterium, Desulfobacterium,Carboxydothermus, Acetogenium, Acetobacterium, Acetoanaerobium,Butyribaceterium and Peptostreptococcus.
 41. The method of claim 35,wherein the carboxydotrophic bacteria is Clostridium autoethanogenum.42. A system for fermentation of a substrate comprising CO, said systemcomprising: at least one bioreactor; determining means adapted todetermine whether a supply of a CO-containing substrate that is providedto a microbial culture is limited or non-limited; and temperaturecontrol means configured such that, in use, the temperature of thebioreactor can be adjusted in response to determination of whether thesupply of the CO-containing substrate for the microbial culture islimited or non-limited.
 43. The system of claim 42, wherein thetemperature control means is configured to reduce the temperature of thebioreactor if the determining means determines that the supply of theCO-containing substrate is limited.
 44. The system of claim 43, whereinthe system further comprises processing means configured such that thetemperature of the bioreactor can be automatically regulated in responseto changes in whether the supply of the CO-containing substrate islimited or non-limited.